In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the specimen. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture stop (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels through the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
Ideally, the track of the scanning light beam on or in the specimen describes a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). At increasingly high beam deflection speeds, the scanning track deviates more and more from the meander shape. This phenomenon is attributable essentially to the inertia of the moving parts. With rapid scanning, the scanning track looks rather like a sine curve, but it often happens that the portion of the track curve for scanning in the positive X direction differs from the portion of the track curve for scanning in the negative X direction.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus sampled one grid point at a time. The measured value must be unequivocally assigned to the associated scan position so that an image can be produced from the measured data.
Advantageously, the status data of the beam deflection device adjustment elements are continuously measured concurrently for this purpose; or, although this is less accurate, the reference control data for the beam deflection device are used directly.
The use of resonantly operating beam deflection devices in order to obtain higher scanning rates is known. In this context, the beam deflection device, which is often embodied as a galvanometer mirror, is operated in a feedback circuit in which a position sensor ascertains the present position of the mirror and converts it into an electrical signal that is then amplified and conveyed to the beam deflection device as the driving signal. This ensures that the beam deflection device is always operated at the resonant frequency, which can fluctuate considerably e.g. as a result of temperature changes.
German Patent Application DE 41 16 387 A1 describes a control system for a laser printer that contains a resonant scanning device having an oscillating mirror which guides the laser beam over the surface of the printing medium in order to illuminate successive pixel locations with the laser beam. Each complete mirror oscillation corresponds to one scanning cycle. A controller serves to pulse the laser source in accordance with a selected image. A synchronization device serves to synchronize the operation of the controller with the angular motion of the mirror. The synchronization device operates continuously during each scanning cycle, and adjusts the operating frequency of the controller, as a function of changes in the angular velocity of the mirror and changes in the resonant frequency of the resonant scanning device, by the fact that a laser pulse timer signal is delivered to the controller.
German Patent Application DE 43 22 694 A1 describes a confocal microscope that contains a scanner arrangement in which the deflection arrangement along the X axis contains two resonant scanners that oscillate about parallel axes at different frequencies, one of which is a harmonic of the other. As a result thereof, scanning along the X axis can be performed almost linearly even though it occurs in conjunction with a resonance, and advantages associated with the rapidity of resonant systems can therefore be achieved. One galvanometer rotates the housing of one of the resonant scanners about its axis in order to achieve an X-axis pivot function.
In order to obtain a defect-free image of the specimen, the time for cycling through one scan line must be a multiple of the time for scanning one specimen point. If this condition is not met, distortion occurs due to image point shifts in successive lines. Since the resonant frequency of the beam deflection device depends on the scanning conditions (for example the maximum deflection of the scanning mirror) and environmental conditions (in particular the temperature), and thus continually fluctuates, whereas the time for scanning a specimen point is constant, with resonant beam deflection devices a faster scanning speed can be obtained only at the expense of image quality.